Tyrosine phosphorylation in semaphorin signalling: shifting into overdrive

The semaphorins constitute a large family of molecular signals with regulatory functions in neuronal development, angiogenesis, cancer progression and immune responses. Accumulating data indicate that semaphorins might trigger multiple signalling pathways, and mediate different and sometimes opposing effects, depending on the cellular context and the particular plexin-associated subunits of the receptor complex, which can include receptor-type or cytoplasmic tyrosine kinases such as MET, ERBB2, VEGFR2, FYN, FES, PYK2 and SRC. It has also been shown that a specific plexin can alternatively associate with different tyrosine kinase receptors, eliciting divergent signalling pathways and functional outcomes. Tyrosine phosphorylation is a pivotal post-translational protein modification that regulates intracellular signalling. Therefore, phosphorylation of tyrosines in the intracellular domain of plexins could determine or modify their interactions with additional signal transducers. Here, we discuss the potential relevance of tyrosine phosphorylation in semaphorin-induced signalling, with an emphasis on its probable role in dictating the choice between multiple pathways and functional outcomes. The identification of implicated tyrosine kinases will pave the way to target individual semaphorin-mediated functions.     

Lysophosphatidic acid modulates the association of PTP1B with N‐cadherin/catenin complex in SKOV3 ovarian cancer cells

LPA (lysophosphatidic acid) is a natural phospholipid that plays important roles in promoting cancer cell proliferation, invasion and metastases. We previously reported that LPA induces ovarian cancer cell dispersal and disruption of AJ (adherens junction) through the activation of SFK (Src family kinases). In this study, we have investigated the regulatory mechanisms during the early phase of LPA‐induced cell dispersal. An in vitro model of the ovarian cancer cell line SKOV3 for cell dispersal was used. LPA induces rapid AJ disruption by increasing the internalization of N‐cadherin‐β‐catenin. By using immunoprecipitations, LPA was shown to induce increased tyrosine phosphorylation of β‐catenin and alter the balance of β‐catenin‐bound SFK and PTP1B (phosphotyrosine phosphatase 1B). The altered balance of tyrosine kinase/phosphatase correlated with a concomitant disintegration of the β‐catenin‐α‐catenin, but not the β‐catenin—N‐cadherin complex. This disintegration of β‐catenin from α‐catenin and the cell dispersal caused by LPA can be rescued by blocking SFK activity with the chemical inhibitor, PP2. More importantly, PP2 also restores the level of PTP1B bound to β‐catenin. We propose that LPA signalling alters AJ stability by changing the dynamics of tyrosine kinase/phosphatase bound to AJ proteins. This work provides further understanding of the early signalling events regulating ovarian cancer cell dispersal and AJ disruption induced by LPA.     

Regulation of CaMKII In vivo: The Importance of Targeting and the Intracellular Microenvironment

CaMKII (calcium/calmodulin-stimulated protein kinase II) is a multifunctional protein kinase that regulates normal neuronal function. CaMKII is regulated by multi-site phosphorylation, which can alter enzyme activity, and targeting to cellular microdomains through interactions with binding proteins. These proteins integrate CaMKII into multiple signalling pathways, which lead to varied functional outcomes following CaMKII phosphorylation, depending on the identity and location of the binding partner. A new phosphorylation site on CaMKII (Thr253) has been identified in vivo. Thr253 phosphorylation controls CaMKII purely by targeting, does not effect enzyme activity, and occurs in response to physiological and pathological stimuli in vivo, but only in CaMKII molecules present in specific cellular locations. This new phosphorylation site offers a potentially novel regulatory mechanism for controlling functional responses elicited by CaMKII that are restricted to specific subcellular locations and/or certain cell types, by controlling interactions with proteins that are expressed in the cell at that location.     

Tyrosine phosphorylation of the human guanylyl cyclase C receptor

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) inEscherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.     

Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.     

Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells

The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.     

Protein-tyrosine phosphatases and the regulation of insulin action.

Protein-tyrosine phosphatases (PTPases) play an important role in the regulation of insulin action by dephosphorylating the active (autophosphorylated) form of the insulin receptor and attenuating its tyrosine kinase activity. PTPases can also modulate post-receptor signalling by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase. Dramatic advances have recently been made in our understanding of PTPases as an extensive family of transmembrane and intracellular proteins that are involved in a number of pathways of cellular signal transduction. Identification of the PTPase(s) which act on various components of the insulin action cascade will not only enhance our understanding of insulin signalling but will also clarify the potential involvement of PTPases in the pathophysiology of insulin-resistant disease states. This brief review provides a summary of reversible tyrosine phosphorylation events in insulin action and available data on candidate PTPases in liver and skeletal muscle that may be involved in the regulation of insulin action.     

Mechanisms of regulating the Raf kinase family

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.     

Thioridazine induces apoptosis by targeting the PI3K/Akt/mTOR pathway in cervical and endometrial cancer cells

Recently, thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methylthiophenothiazine), a well-known anti-psychotic agent was found to have anti-cancer activity in cancer cells. However, the molecular mechanism of the agent in cellular signal pathways has not been well defined. Thioridazine significantly increased early- and late-stage apoptotic fraction in cervical and endometrial cancer cells, suggesting that suppression of cell growth by thioridazine was due to the induction of apoptosis. Cell cycle analysis indicated thioridazine induced the down-regulation of cyclin D1, cyclin A and CDK4, and the induction of p21 and p27, a cyclin-dependent kinase inhibitor. Additionally, we compared the influence of thioridazine with cisplatin used as a control, and similar patterns between the two drugs were observed in cervical and endometrial cancer cell lines. Furthermore, as expected, thioridazine successfully inhibited phosphorylation of Akt, phosphorylation of 4E-BP1 and phosphorylation of p70S6K, which is one of the best characterized targets of the mTOR complex cascade. These results suggest that thioridazine effectively suppresses tumor growth activity by targeting the PI3K/Akt/mTOR/p70S6K signaling pathway.     

AMP-activated protein kinase--the key role in metabolic regulation

AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that acts as an energy sensor maintaining the energy balance at the cellular as well as at the whole body level. Within the healthy cell, metabolic stress leading to an increase in AMP concentration results in AMPK activation. Once activated, AMPK "switches off" many anabolic pathways e.g. fatty acid and protein synthesis while "switches on" catabolic pathways such as fatty acid oxidation or glycolysis which serve to restore intracellular ATP level. Adipocyte derived hormones leptin and adiponectin activate AMPK in peripheral tissues increasing energy expenditure. AMPK also regulates food intake due to response to hormonal and nutrient signals in hypothalamus. Antidiabetic drugs that mimic the action of insulin activate the AMPK signaling pathways. Further studies are needed to clarify the importance of the AMPK activation for therapeutic effects of this drugs.     

AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade

Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities. Processive relay of signals through RAF-MEK-ERK modulates cell growth and proliferation. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838 on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events.     

c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs

The human pathogen Helicobacter pylori colonizes the mucous layer of the stomach. During parasitic infection, freely swimming bacteria adhere to the gastric epithelial cells and trigger intracellular signalling pathways. This process requires the translocation of the effector protein CagA into the host cell through a specialized type IV secretion system encoded in the cag pathogenicity island. Following transfer, CagA is phosphorylated on tyrosine residues by a host cell kinase. Here, we describe how the tyrosine phosphorylation of CagA is restricted to a previously identified repeated sequence called D1. This sequence is located in the C-terminal half of the protein and contains the five-amino-acid motif EPIYA, which is amplified by duplications in a large fraction of clinical isolates. Tyrosine phosphorylation of CagA is essential for the activation process that leads to dramatic changes in the morphology of cells growing in culture. In addition, we observed that two members of the src kinases family, c-Src and Lyn, account for most of the CagA-specific kinase activity in host cell lysates. Thus, CagA translocation followed by tyrosine phosphorylation at the EPIYA motifs promotes a growth factor-like response with intense cytoskeletal rearrangements, cell elongation effects and increased cellular motility.     

Control of the eukaryotic cell cycle by MAP kinase signaling pathways.

In an often rapidly changing environment, cells must adapt by monitoring and reacting quickly to extracellular stimuli detected by membrane-bound receptors and proteins. Reversible phosphorylation of intracellular regulatory proteins has emerged as a crucial mechanism effecting the transmission and modulation of such signals and is determined by the relative activities of protein kinases and phosphatases within the cell. These are often arranged into complex signaling networks that may function independently or be subject to cross-regulation. Recently, genetic and biochemical analyses have identified the universally conserved mitogen-activated protein (MAP) kinase cascade as one of the most ubiquitous signal transduction systems. This pathway is activated after a variety of cellular stimuli and regulates numerous physiological processes, particularly the cell division cycle. Progression through the cell cycle is critically dependent on the presence of environmental growth factors and stress stimuli, and failure to correctly integrate such signals into the cell cycle machinery can lead to the accumulation of genetic damage and genomic instability characteristic of cancer cells. Here we focus on the MAP kinase cascade and discuss the molecular mechanisms by which these extensively studied signaling pathways influence cell growth and proliferation.     

Phosphorylation-mediated activation and translocation of the cyclic AMP-specific phosphodiesterase PDE4D3 by cyclic AMP-dependent protein kinase and mitogen-activated protein kinases. A potential mechanism allowing for the coordinated regulation of PDE4D activity and targeting

In this study, we describe a novel mechanism by which a protein kinase C (PKC)-mediated activation of the Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade regulates the activity and membrane targeting of members of the cyclic AMP-specific phosphodiesterase D family (PDE4D). Using a combination of pharmacological and biochemical approaches, we show that increases in intracellular cAMP cause a protein kinase A-mediated phosphorylation and activation of the two PDE4D variants expressed in vascular smooth muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that stimulation of PKC via the associated activation of the Raf-MEK-ERK cascade results in the phosphorylation and activation of PDE4D3 in these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP.     

Tyrosine phosphorylation enhances the SH2 domain-binding activity of Bcr and inhibits Bcr interaction with 14-3-3 proteins.

The cellular Bcr protein consists of an N-terminal serine/threonine kinase domain, a central guanine nucleotide exchange factor homology region and a C-terminal GTPase-activating protein domain. Previous work in our laboratory established that Bcr is a major transformation-related substrate for the v-Fps tyrosine kinase, and tyrosine phosphorylation of Bcr induces Bcr-Grb-2/SOS association in vivo through the Src homology 2 (SH2) domain of Grb-2. In the present study, we mapped the region of Bcr tyrosine phosphorylation by c-Fes, the human homologue of v-Fps, to Bcr N-terminal amino acids 162-413 by using a baculovirus/Sf-9 cell co-expression system. Tyrosine phosphorylation of Bcr by Fes greatly enhanced the binding of Bcr to the SH2 domains of multiple signalling molecules in vitro, including Grb-2, Ras GTPase activating protein, phospholipase C-gamma, the 85,000 M(r) subunit of phosphatidylinositol 3'-kinase, and the Abl tyrosine kinase. In contrast with SH2 binding, tyrosine phosphorylation of Bcr reduced its ability to associate with the 14-3-3 protein Bap-1 (Bcr-associated protein-1), a Bcr substrate and member of a family of phosphoserine-binding adaptor proteins. These experiments provide in vitro evidence that tyrosine phosphorylation may modulate the interaction of Bcr with multiple growth-regulatory signalling pathways.     

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.     

Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion

Cadherins are homophilic cell–cell adhesion molecules implicated in many fundamental processes, such as morphogenesis, cell growth, and differentiation. They accumulate at cell–cell contact sites and assemble into large macromolecular complexes named adherens junctions (AJs). Cadherin targeting and function are regulated by various cellular processes, many players of which remain to be uncovered. Here we identify the small GTPase Rab35 as a new regulator of cadherin trafficking and stabilization at cell–cell contacts in C2C12 myoblasts and HeLa cells. We find that Rab35 accumulates at cell–cell contacts in a cadherin-dependent manner. Knockdown of Rab35 or expression of a dominant-negative form of Rab35 impaired N- and M-cadherin recruitment to cell–cell contacts, their stabilization at the plasma membrane, and association with p120 catenin and led to their accumulation in transferrin-, clathrin-, and AP-2–positive intracellular vesicles. We also find that Rab35 function is required for PIP5KIγ accumulation at cell–cell contacts and phosphatidyl inositol 4,5-bisphosphate production, which is involved in cadherin stabilization at contact sites. Finally, we show that Rab35 regulates myoblast fusion, a major cellular process under the control of cadherin-dependent signaling. Taken together, these results reveal that Rab35 regulates cadherin-dependent AJ formation and myoblast fusion.